Bsa c

For immunohistochemical studies, hearts were snap frozen in liquid nitrogen, or frozen in isopentane at −50°C, or incubated overnight in 4% PFA followed by overnight incubations in 20% and 30% sucrose in PBS, prior to embedding in OCT and storage at −80°C. 10–15 µm sections were cut using a cryostat (Bright instruments), air dried and immersed in 4% PFA in PBS or in acetone at −20°C for 15 min and washed for 3×5 min in 0.1% PBS-Triton X-100. Blocking was achieved by incubation with 5% BSA-C (Aurion) in 0.1% PBS-Triton X-100 for at least 30 min at RT. Immunolabeling with primary antibodies was performed in 0.1% PBS-Triton X-100, 1% BSA-C overnight in a humidity box at 4°C as described previously [15] (link). Sections were washed 3× in PBS, incubated for 60 min at RT in a dark box with the anti-rabbit (FITC Invitrogen 1∶1000 in PBS), washed 3× in PBS and counterstained with DAPI (Invitrogen). Sections were mounted in Vectashield mounting medium (Vector Laboratories). Sections were examined using the Leica TCS SP4 laser scanning confocal microscope and analysed with Leica Application Suite (LAS) v5 (Leica Microsystems, Heidelberg, Germany).

Cryoimmuno-EM labelling was performed as previously described67 (link)68 (link). Briefly, cells were fixed either in 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The fixed cells were embedded in 10% gelatin and small blocks were infused with 2.3 M sucrose overnight and then frozen in liquid nitrogen. Ultrathin cryosections (50 nm) were cut at −120 °C with a cryo-ultramicrotome (UCT7, Leica, Vienna, Austria). The goat polyclonal antibody recognizing IGF2 (sc-7435, diluted 1:10) and protein G gold (Aurion, the Netherlands) that detect primary polyclonal antibody were used. All antibodies and gold conjugates were diluted in 0.1% BSA-c (Aurion, the Netherlands) in PBS.

H7N9 VLPs (4 μL; 300 ng/μL) were first incubated on a copper EM grid for 2 minutes. The grid was blocked in a 40 μL drop of 1X DPBS supplemented with 5% BSA (AURION BSA-c, 900.099) and 0.1% cold water fish skin gelatin (AURION, 900.033). Following a 30-minute incubation in a glass petri dish incubation chamber, the grid was stained in a 40 μL drop of rabbit polyclonal anti-HA7 antibody (Sino Biological Inc., 40103-RP02, 1: 500 dilution in blocking buffer) or blocking buffer alone to serve as a negative control. The grid was placed in a 40 μL drop of secondary antibody (Donkey-anti Rabbit IgG, Gold 10nm particle size, Electron Microscopy Sciences, 2705; diluted 1:40 in blocking buffer). The grids were stained with 2% uranyl acetate and observed using a 200kV JEOL 2100 scanning and transmission electron microscope (Japan Electron Optics Laboratories). Images were captured using a high-resolution digital camera (U-1000, www.gatan.com).

Freshly isolated NK cells or CTL, resting or stimulated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Calbiochem) and 0.5 mM ionomycin (Sigma-Aldrich), or co-incubated with K562 cells were spun down in glass-bottom plates (Matrical, Brooks). After 15 min, cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% saponin in PBS, and blocked in PBS with 5% FBS, 0.1% BSA-c (Aurion), and 2% normal donkey serum (Jackson ImmunoResearch). Images were acquired on a confocal microscope (Nikon A1R) with a 63× oil objective, using NIS Elements Software, and analyzed using ImageJ (Research Service Branch, National Institutes of Health, Bethesda, MD) or PAD (Digital Cell Imaging Laboratories, Belgium). To quantify the size and number of lytic granules, mean fluorescence intensity, as well as distances between granules, immune synapses (IS) and the microtubule-organizing center (MTOC), images of fixed cells with were analyzed using the PAD software. Object detection based on perforin or CD107a labeling was performed automatically and checked manually. MTOC XY co-ordinates were selected manually based on α-tubulin labeling, and the center of the IS was entered based on phalloidin labeling and phase contrast images. Mean and standard deviation (SD) per patient were then graphed using Prism.

Aliquots of suprastructural fragments were absorbed to formvar/carbon nickel grids, washed with PBS and treated for 30 min with 2% (w/v) dried skim milk in PBS for cartilage and bone or 2% (w/v) bovine serum albumin (BSA) in PBS for skin. This procedure was followed by incubation with recombinant Emp (full-length or fragments) in PBS at different concentrations for 1 h at RT. After several steps of washing with PBS, the grids were allowed to react with a rabbit polyclonal antibody against Emp diluted 1:100 in 0.2% (w/v) dried skim milk or PBS containing 2% (v/v) BSA-c (Aurion, Wageningen, Netherland) and 0.025% (v/v) Tween 20 (blocking solution). After washing with PBS, the grids were placed on drops of PBS containing goat antibodies against rabbit immunoglobulins conjugated to colloidal gold particles (Jackson Immuno Research Laboratories) diluted 1:30 in 0.2% dried skim milk or blocking solution. In some experiments, the suprastructural fragments were treated with purified collagenase (CLSPA; Worthington, New Jersey, USA) for 2 h at 37 °C. Finally, the grids were washed five times with distilled water and negatively stained with 2% (w/v) uranyl acetate for 10 min. Electron micrographs were obtained with a Philips EM-410 electron microscope at 60 kV.

The T8100 sections from all four eyes were pretreated with 0.05% trypsin (Gibco, Paisley, Scotland) in Tris buffer (pH 7.8) containing 0.1% CaCl₂, for 15 minutes at 37°C, for antigen retrieval. The sections were washed in phosphate buffer and incubated in 0.1 M citric acid pH 3.0 for 30 minutes at 37°C and washed. In order to block nonspecific binding of the primary antibodies, sections were incubated with PBS containing 2% BSA and 2% serum for 30 minutes at room temperature. The primary antibodies were diluted in phosphate buffer/1% BSA-c (Aurion, Wageningen, the Netherlands) (1:100). Sections were incubated in primary antibody initially for 2 hours at 37°C and then at room temperature overnight. Primary antibodies included the rabbit polyclonal and mouse monoclonals against Col VII. Sections were incubated in SARPO or RAMPO (1:500) in PBS with 1% BSA and 2% human serum for 1 hour. Sections were stained with 3-amino-9-ethylcarbazole and counterstained with hematoxylin. In control sections primary antibody was omitted.

The above described ultrathin sections of transduced HepG2 cells embedded in LR White were incubated for 20 min with 50 mM Glycine before blocking for 30 min with blocking buffer (0.5% BSA/0.1% gelatin (Cold Water Fish Skin, EMS, Hatfield, PA, USA)) followed by 5 min incubation in 0.1% acetylated BSA (BSA-c) (Aurion, Wageningen, The Netherlands), pH 7.5 at room temperature (RT). Incubation was done overnight with the polyclonal goat anti-RV serum (obtained from Catherine Eichwald, University of Zurich, Zurich, Switzerland) diluted 1:10 in 0.1% BSA-c at 4 °C. After washing five times with BSA-c, the sections were incubated with the secondary anti-goat antibody conjugated with Alexa Fluor 488 (Molecular Probes), diluted 1:500 in 0.1% BSA-c for 1 h at RT. After washing 5 times with 0.1% BSA-c and once with H₂O, the sections were stained with DAPI (0.1 mg/mL) for 15 min followed by 2 washes with PBS. After the staining, the sections were sandwiched between a microscope slide and a coverslip in PBS and sealed with nail polish. With a fluorescence microscope (Axio Observer inverted microscope), the whole grid was imaged in bright field and fluorescence and several images were taken with a 100× oil (NA = 1.25) objective. These images served as maps and were then used to find the same areas in the scanning electron microscope (SEM).